A DEMGN‘STRATI‘GR OF FLUORESCENT ANTIBODY STAINIKG T0 LOCALKZE ENBGGENDUS JUVENILE HGEMQREvUKE WCULES EN SELECTED TiSSUES (IF THE AMERICAH CQCKBGACH. PERIPLANEIA AMERICANA A Disurfahon {or Hm Dogma of pin. D. MlCfliGAE STATE UNEVEKSITY Howard Dougi‘as Booth 1974 Michigan Stan: University This is to certify that the _ thesis entitled .. ~ _.3 '8’ A DEMONSTRATION OF FLUORESCENT ANTIBODY STAINING TO LOCALIZE ENDOGENOUS JUVENILE HORMONE-LIKE MOLECULES IN SELECTED TISSUES OF THE AMERICAN COCKROACH, PERIPLANETA AMERICANA presented by Howard Douglas Booth has been accepted towards fulfillment of the requirements for Ph. D. degree in Entomology flag“ WMMdV xi Major professor ‘/ Date J’/'7r% 0-7639 LIBRARY “ ‘ .‘,,a.,4~o’o—O4fir- _il ‘2 Av epoxy FA fraction #2 0.5 ul ] ABAAASlIlo Gas Liquid Chromatograph Data Sheet (10/4/72-1) 1. Figure .A~-~a\e\oav poonm open nannmopaeouso unsung new .m enemas 34 o ASHE mom fl :HV IH 1N A rd mm A urn 1w Min .m _u d mic m c t s u a-.. A : e O O F 1 t 3 1 3 3 Tea L: I. 35 .Amn\w\mv swashoosox mm copmwsnsoo Sony couzaomuzm mm no voenm span :QQOOHMEonno casuag moo .m enamwm 1(l()l(/{/)/l()l() Anwa m n :Hv 13:40 3 x 10‘11 4:40 3:40 1.0 pl injection. 32x conc. hydrolysis. 0.5 ul FA standard 1 ul/ml 3 x 10'11 2:35 36 The earliest appearance of a peak in the FA region occurred at the 6x concentration of the sample with a detector voltage setting of l x 10—10 AFS. 11 AFS attenuation At 32x concentration and 3 x 10' three distinct peaks were evident. The first peak at 3:40 min had a 13 mm height and a 2 mm base. The second at 4:40 min had a 7.5 mm height and a 3 mm base. The third peak at 13:30 min with a 13 mm height and an 8 mm base was out of the typical FA range. but could quite possibly represent hydrolysis degradation pro- ducts from the epoxy FA. such as closed ring molecules. This possibility is supported by an earlier test hydrolysis of a sample of FA in which a large peak was also obtained in the 13:30 min area. The quantities were calculated from peak area of the known FA standard concentration (0.88 pg/ml) with a 0.5 pl injection. The combined peak area of 266 mm2 was divided into the 0.44 pg injection and yielded .0017 pg/mmz. The hydrolysis extract peaks were then quantified and divided by the concentration factor (64x) to yield the total micrograms of each in the original 3.0 m1 sample from a 1.0 m1 starting sample. The results are shown in Table 2. 37 Table 2. Hydrolyzed Farnesenic Acid Products. us/ (iii? = mm2 := pg/ml or extract sample 3:40 13.00 0.345 or 100” 4:40 11.25 00299 or 0089 13:40 52.00 1.380 or 4.14 Thus. from a starting 1.0 m1 of conjugated hemocyanin epoxy FA (1.2 nM). at least 1.94 pg (peaks 3:40 and 4:40 min) and probably 6.04 pg (total peaks) of the epoxy FA products were recovered. The figure. 1.94 pg divided by .252 pg/nM yields 7.68 nM of FA. This. divided by the 1.2 nM of the hemocyanin would show at the minimum an average of about 6.4 molecules of epoxy FA to be conjugated to a molecule of hemocyanin. More likely. the 6.04 pg (23.9 nM) yielding 20 molecules of FA per molecule of hemocyanin is a more accurate estimate. In either case. this demonstrated a minimally (to moderately. ca. 20) adequate uptake of the epoxy FA to predict probable success in stimula- tion of hapten antibody formation. 38 III. Antibody Titer and Testing Results from Equilibrium Dialysis Radioimmunoassay (EDRA). Initial titer testing using a solid phase radio- immunoassay was attempted. This technique involved coating a polyethylene tube with an antibody mono- layer to detect selective uptake of an isotopically labelled antigen (Peron and Caldwell. 1970. pp. 87-147). After eight weeks and six experimental runs incor- porating a diversity of purification and design variation steps. this method was rejected since most trials yielded inconclusive results. The major problem areas seemed to be low titer levels. antibody purification. and low isotope specific activity for this type of assay. Standard alpha and beta precipitation tests and ring tests were run to demonstrate the presence of hemocyanin antibody as would be expected. Early attempts to detect anti-juvenile hormone via these tests did not yield positive results. Changing to equilibrium dialysis radioimmunoassay (EDRA) as a method to quantify anti JH allowed the use of much larger quantities of both sera and isotope. This resulted in the detection and quantification of positive anti JH titers. To equate the titer testing data over a 53 month period when EDRA's were being run. molar antigen 39 uptake equivalents of the anti JH (Ab) were calcu- lated and the results are shown in Table 3. The net CPM from the average normal serum control (NS) was subtracted from the average net anti JH CPM samples. thus yielding the net selective binding (NSB) CPM. This was divided by 0.90 ml (sample volume counted) to give NSB cpm/n1. The NSB CPM were divided by 71% efficiency of the counting system to yield the NSB DPM/ml. This. in turn. was divided by the specific activity of the methyl juvenate 1“0 (MJ 1“0) at 140 pCi/mM (= 1170 DPM/pg) to give the weight of antigen (MJ 1”0) taken up by the 1 mg/ml Ig solution con- taining the antibodies. The nanogram quantities of MJ 1“0 were divided by 266 ng/nM of methyl juvenate to give the molar uptake of antigen. In a reaction with generous excess of antigen the antibodies (primarily IgG class) will bind with two molecules of antigen per molecule of antibody so the halving of the nanomolar antigen would give the nanomolar equivalent of antibody involved in antigen binding. The molar antibody concentration multiplied by 166000 ng/nM of IgG yielded the nanograms of anti JH. This was plotted as the pg of anti JH per 1.00 ml solution of 1 mg/Ml serum Ig against two week time intervals. The graph in Figure 4. shows the titer 4O .Ha\ms o.H as o.oo u mmz o.ms er .enor cospaonmsnsa snaoeooom * o.mrm Aas\ms o.Hv Aom\Hvs< .o>< .hmmmmocssawowumm mfimzamfin sawmpflaflsvm mo mpasmcm nopwa .m manna L... 41 .mpcoam>wsum oprmp somapc< mmaos .3 opswwm Amm\$m\mv povmoom vmmn u + NN 0N ma 0H .aa NH 0H m o d N mxooz _ _ _ F _ p _ a _ a 8 Cl], .I. m {NH // o~o¢///// v \ /// 10H u III n+ Ill/I T: as} / M / I ma ) / n / fl / w / 1. ON [TU / ‘1‘): O .\ S n as a \ 1mm 42 levels and the expected decline after booster injections were stopped. The titer data illustrates specific antibody levels from 4.6 to 22.1 pg of anti JH per mg of Ig protein. thus making up from 0.46% to 2.09% of the total protein. In a test of further purification DEAE cellulose ion exchange was used to isolate the IgG fraction from the Ig sera (Williams and Chase. 1967. Vol. 1.. pp 322-326). A 0.75 mg/ml IgG fraction yielded a molar antigen binding equivalent to 18.8 pg/ml (or 25.0 pg/Mg). Thus. purification concentrated the antibody from 2.09% total protein to 2.51% in a 1.0 mg/ml solution (see Table 3. 7/19/73*). IV. Fluorescent Antibody Staining Results. Initial photography using color film was generally less satisfactory than black and white. due to the fluorescence fading during film exposure along with somewhat reduced contrast and higher cost of materials. After initial trials of staining with serum dilutions from 10.0 to 0.01 mg/ml Ig. observa- tions and photographs were taken of slides stained with 1.0 to 10.0 mg/ml Ig. Photographic data is further delineated in Table 4 and Figures 5 to 19 show the aetual photographic results. “3 In the first series of photos (Fig. 5 and 6) Ab and NS demonstrated good contrast and specific cyto- plasmic fluorescence in the corpra allata tissue area of whole brain sections 6 p thick. The Ab and NS photos cannot be directly compared in this set because the longer exposure time of Ab (Fig. 5) increased the fluorescence intensity somewhat. However. the presence of specific fluorescence is evident. The next series (Fig. 7. 8 and 9) was of whole mount corpra allata stained and photographed at 250x with 3.5 sec exposure time. A non-stained control was added to the series. Both the normal serum control (Fig. 8) and the non-stained (Fig. 9) show markedly reduced cytoplasmic fluorescence when contrasted to the antibody stained (Fig. 7). Note that the brightly fluorescing rectangular structure on the upper surface of Fig. 7 is a piece of tracheal intensely auto- fluorescing. With improving cryostat technique. sections containing only corpra allata were cut at 3 p thick- ness. Sections of Ab. NS and 00 (Fig. 10. 11 and 12) were stained with more dilute sera (1.0 mg/mD and more dilute FITC antiglobulin 1:20 in an attempt to diminish the nonspecific fluorescence. EXposure time was increased to 5 sec and photos were taken at 540x. 4b Again. in closely comparable sections. somewhat greater cytoplasmic fluorescence is evident in Fig. 10 with similar and diminished fluorescence in the con- trols (Fig. 11 and 12). Using another slide stained under the same conditions, photos (Fig. 13 and 14) were taken at 950x with only UV incident light. Exposure time was increased to 10 sec for each. While the overall intensity was diminished by the use of only UV light. the contrast between Ab and NS control (Fig. 13 and 1h) was enhanced. Hemocytes had been implicated as a likely area for early concentration of exogenous isotopically labelled juvenile hormone (Emmerick. 1973) so hemocyte smears were prepared and stained with the 10 mg/ml sera and 1:10 FITC-antiglobulin photographs were taken at 590x with blue and UV incident light. Antibody stained hemocytes (Fig. 15 to 19) showed distinct cytoplasmic fluorescence at 5 sec exposure. Normal serum stained control showed no fluorescence at 5 see (Fig. 16). only a most faint blur at 20 sec (Fig. 17). and a more pronounced blur at #0 sec (Fig. 18). The non-stained control (Fig. 19) hemocytes showed blurred images at 50 sec exposure time. The reason for this much greater contrast in hemocyte antibody staining as compared to the results 45 of the corpra allata whole and sectioned staining await further investigation. Yet. in both tissues a difference in the binding of the fluorescence iso- thiocyanate molecule via an anti rabbit globulin antibody, to an attached rabbit antibody, to an insect material was selectively demonstrable. 46 xOOO 0.0m oan O >O Ocoz Ocoz semen mesoosom HOOVOO-NN\~Hum OH erO 0.0e OsHO e >O OOHO OH.H mz He\ws OH “seem opsoosmm AOsvmznmm\NHnm OH xOsO 0.0m OOHO O >O OOHm OH.H mz Hs\me OH names mes0020m Homvmzum~\~Hnm 5H xosm 0.0 OOHO O >O OaHm OH.H mz Hest OH paces mphoogmm Amvmzumm\~Hum OH xOOO 0.0 msHO e >O OeHm OH.H p< Hs\ms OH names messages HOVOO-NN\~Hum OH xOOO O.OH cho >O OOHO O~.H mz H5\ws H an epsHHw euOuoo nomom mz-OH\H->O 2H NOOO O.OH cho >O OOHO ON.H O< He\we H an semHHm seduce noses O<-OH\H->O mH «OOO 0.0 OOHO O >O meoz 0:02 1m speHHs seduce nomom OO-OH\H NH eoem O.O oan e >O OBHO O~.H mz H5\we H 1m spmHHs seduce cosom mzuOH\H HH xOsO 0.0 osHp e >O OOHO O~.H O< Hs\me H an epmHHm seduce gowom OeuOH\H OH xOON O.m oaHn O >: Ocoz Ocoz s; upmHHs «yahoo nomom OO1~N\~H O xOmm O.m OOHO O >O OOHO OH.H mz H5\ms OH a; mesHHs seduce noses mz-NN\NH O xOON O.m OOHO O >O OOHO OH.H ne H2\ws OH 2: spsHHs seduce :osom nO OOHm OH.H mz Hexme OH 1 O cHsuO noses mz-mo\HH O xOOO O.: man e >O OOHO OH.H pe stws OH 3 O :Hmun comes OeuOO\HH O Aommv pnmfla * .wmz mafia Pcou caspm whom osmwwa ovoo opozm .mH .mxm swosH .m .mpasmom mswcflmpm mvopfipc< pcmomouosam how even owgmmpwoposm .e OHnsa “7 Figure 5. Antibody Roach Brain, 6 u Vand blue light, # sec, 5h0x mag. 48 Figure 6. Normal Serum Roach Brain. 6 u UV and blue light. 3 sec. 540x mag. 49 “a Figure 7. Antibody Roach Corpra Allata. wm. UV and blue light. 3. 5 sec. 250x mag. 50 Figure 8. Normal Serum Roach Corpra Allata. wm. UV and blue light, 3.5 sec, 250x mag. 51 Figure 9. Stain Only Roach Corpra Allata. wm. Uv and blue light. 3.5 sec. 250x mag. 52 Figure 10. Antibody Roach Corpra Allata. 3 u UV and blue light. 5 sec, 5h0x mag. 53 Figure 11. Normal Serum Roach Corpra Allata, 3 u UV and blue light. 5 sec, 5#0x mag. 5# Figure 12. No Stain Roach Corpra Allata, 3 u UV and blue light. 5 sec. 5#0x mag. 55 Figure 13. Antibody Roach Corpra Allata. 3 p. UV light only. 10 sec. 950x mag. 56 Figure 14. Normal Serum Roach Corpra Allata, 3 u UV light only. 10 sec. 950x mag. 57 Figure 15. Antibody Hemocyte Smear UV and blue light. 5 sec, 5#0x mag. 58 Figure 16. Normal Serum Hemocyte Smear UV and blue light. 5 sec, 51i0x mag. 59 _, a... H ’~_I-“ RV. '— - --.-.. .- Figure 17. Normal Serum Hemocyte Smear UV and blue light. 20 sec, 540x mag. 60 Figure 18. Normal Serum Hemocyte Smear UV and blue light, #0 sec, 590x mag. (Note the arrow indicating an artifact.) 61 Figure 19. No Stain Hemocyte Smear UV and blue light. 50 sec, 5#Ox mag. DISCUSSION Before the actual research to make an antibody against epoxy FA could start, it was necessary to answer two preliminary feasibility questions: First. acting on the unlikely possibility that rabbit serum contains naturally occurring antibodies that would selectively bind a juvenile hormone-like molecule, normal serum was tested for methyl juvenate uptake using methods similar to those described in the Equilbrium Dialysis Radioimmunoassay section. The normal serum was shown to have only low levels of uptake which should not interfere with the immuniza- tion procedure. Secondly. the small size of the proposed antigen, epoxy FA (266 daltons). would very likely require a large protein carrier molecule if an immune response was to be stimulated. The absence of amino groups on the epoxy FA hapten required the adoption of a less commonly used conjugation technique. The reaction makes use of a carbodiimide to link the epoxy FA carboxyl to one of the hemocyanin amino acid units which has an available amino group (2,3,, lycine- aspergine). Before investing the time and money into 62 63 synthesis and purification of the epoxy FA a pre- liminary conjugation and subsequent hydrolysis reaction using FA was completed using the procedure described for the epoxy FA. This also gave positive results that deemed further investigation feasible. The choice of epoxy FA as the juvenile hormone analogue to use as a haptenic source of antibody was based on its relatively close molecular configuration to naturally occurring Cecropia juvenile hormone (C-18) trans trans cis methyl lO-ll epoxy-7-ethyl 3.11 dimethyl 2.6-tridecadienoate (Roller. 196?) and its closer similarity to the likely naturally occurring (C-l6) methyl lO-ll epoxy 3.7.11 trimethyl 2.6-dodecadienoate. The C-l6 has been demonstrated as a probable corpra allata synthesis product based on work with Orthopterans and Lepidopterans (Riddiford and Ajami. 1973 and Judy. 1973). The available isotopic source for radioimmuno- logical testing was MJ luc (lO-ll epoxy methyl farnesoate) which has the same molecular configuration except for the methyl ester which is located in a homologous position to the carrier attachment site on the epoxy FA molecule. Therefore. it would be expected to contribute very little. if any. to the antibody-antigen binding reaction (Boyd. 1962). The 6h epoxy FA molecule has the necessary carboxyl group available for participation in the carbodiimide con- jugation reaction. The carbodiimide conjugation reaction was modified from a procedure for protein to protein linking via carboxyl groups (Williams and Chase. 1967. pp. 155-158). The epoxy FA carboxyl reacted with a carbodi- imide which undergoes rearrangement. then couples with a second molecule of epoxy FA to form the acid anhydride. This. in turn. combines with an available amino group from the hemocyanin chain to form a peptide bond and releases the second epoxy FA molecule (see Figure 20). The immunization procedure designed was a modification of that used by Borst to make ecdysone antisera (Borst. 1972). The procedure used was one which would give the maximum opportunity for antibody formation through secondary immune response (IR) enhancement of a booster series. To further enhance the IR. the hapten-carrier conjugate was injected with an adjuvant in which attenuated bacteria heighten IR sensitivity and the oil emulsification of the antigen prolong its metabolic availability. Subcutaneous injection and a non-antigenic booster medium were utilized to minimize the hazard of rabbit loss to 65 .sHsmhoosom op coapmwsncoo Sm Enema .om shaman 4 _ .opm .ockummmm .9223 ..M...ml .msosw 2. 958m 0.3.3.328 59H: mufio< I ocws< .chmhooEOI I I I I .0" O v IuZuUUUUI M = _ _ IOOI+ OIIIIIZI 322222 N MOO 32 I.u QIU-OIUI.& . . OOHEHHconpmo HzxosoaomOHn o a :2 PCmEmmcmuHMWV 2\ \U0 U ad All 0 2| InU Z . .+ IOOUIN. ‘ 2.92 . Q I O I CE @8le u 58-5 )v 66 infection or anaphylactic shock. For this same reason. ear bleeding for serum harvest was chosen in place of the more rapid. but more hazardous. cardiac puncture. Classical titer testing methods such as preci- pitation. hemagglutination or hemolysis were bypassed because the anti JH titer levels were expected to be very low. and both the radioisotopic antigen and scintillation counting equipment provided a more sensitive quantitative assay technique. For initial titer testing a solid phase radioimmunoassay pro- cedure was designed which has the promise of high sensitivity and low volume use of a sparse radio- isotopic antigen supply (Peron and Caldwell. 1970). Unfortunately. this did not produce significant results. A greater time- and isotope-consuming equili- brium dialysis radioimmunoassay was designed. following the general procedures outlined in Weir (1967). This was possible because the small molecular size of the antigen (MJ 14C) allowed it to pass freely through the dialysis membrane while the serum proteins could not. This free flow was confirmed in buffer control samples. Because the biologically active half life of the synthesized MJ lac was around six to twelve 67 months (personal communication) some isotopically labelled degradation products would be in the antigen solution. While further isolation and testing of these products would be required to confirm their antigenic .1 '1. activity. it was assumed in this research that selective uptake would be almost entirely by those molecules retaining their bioactive configurations. because in any reaction there would be a massive _m‘mmm *‘nll‘tfirc- “i. vv'fi'li excess of available antigen molecules for any one site. Thus. if a specificity preference is demonstrated. there would be a strong tendency to establish an equilibrium in which the most site-compatible molecules would be found in those sites. The other. if binding at all. would do so less selectively. Even if there was selective binding of some of the degradation molecules. for the purpose of this research. it could still represent an antibody binding site specific to only a part of the antigen molecule. Hence. it would add to the titer level a small cross-reaction factor. but should not significantly change it. Corpra allata and hemolymph were selected from adult cockroaches (in most cases. males) at least two weeks past their last moult. Based on JH bioassays of corpra allata and hemolymph in Locusta and 68 §chi§tocerca (Johnson. 1973) as well as the less reliable corpra allata histology-activity estimates of Leucophaea (Scharrer. 1958). this would be sufficient time to expect a return to higher and relatively constant endogenous JH production. Rapid dissection and immediate freezing were employed to minimize any enzymatic degradation of the JH. Cryostat cut frozen sectioning and direct mount- ing made it convenient to avoid the organic reagents (i,g,. ethanol. xylene. paraffin) which would most probably dissolve the tissue JH and remove the antigen source for staining. An indirect fluorescent antibody staining (IRAS) technique was used in which an antibody (immunoglobulin) is first bound to an antigen in the tissue. and then an antiglobulin which has the fluorochrome attached is reacted to bind with the antibody molecule. This results secondarily in having several fluorescing tags attached to the site of the antigen. This indirect method has the advantages of: 1) allowing the use of standardized commercial fluorochrome tagged antiglobulin solutions at a considerable saving of time. and 2) enhancing the fluorescence of the antigen site by allowing the attachment of several fluorochrome-bearing antiglobulin molecules rather 69 than a single antibody molecule as with direct fluorescent antibody staining. But any fluorescent antibody staining method and especially the IFAS. requires precise controls to interpret the resultant fluorescence. In the preparations of reagents. protein concen- trations of antisera and normal sera were equated to keep the normal occurrence of some nonspecific protein binding the same. Any nonspecific protein bound in the initial incubation would bind antiglobulin molecules at the staining step and appear to be tissue antigens. It was also necessary to have complete washing of the tissues after both incubation and staining steps to remove as much of the nonbound proteins as is possible. Additional controls treated with only stain or only normal serum were incorporated to quantify the resultant nonspecific fluorescence. Placement of the controls on the same coverglass assured equal treatment at this step. Another source of nonantigen related fluorescence is autofluorescence. Autofluorescence of various tissue components (some proteins. nucleic acids. etc.) was taken into consideration by the inclusion of a control that was treated with neither serum nor stain. For the hemocyte staining a second test area was 70 included to compare the repeatability of the staining from one section to another in the same series. To maximize the contrast. both sera and stain solutions were tested through serial dilution to find the optimum level of maintained specific fluor- escence with nonspecific fluorescence at a minimum. This resulted in a three way trade-off situation in which the minimal total light requirement took precedence in order to have low photograph exposure times. thereby limiting the fluorescent fading effect. Within these limiting factors the level of optimum contrast for photography was somewhat less than that for visual observation. but correlated through the range. With autofluorescence varying in different tissues it was also necessary to match the histology of test and control tissues carefully when working with multi-tissue sections. This was much reduced after sections of only corpra allata or only hemolymph were utilized. Finally. the focus of incident light often requires adjustment when changing slide or magnifi- cation levels. easily introducing intensity error. This was offset by placement of test and control 71 tissues on the same slide which facilitated com- parisons at the same magnification without alteration of light adjustment between sections. CONCLUSIONS In conclusion. it was found that dicyclohexyl carbodiimide reacting with epoxy farnesenic acid and keyhole limpet hemocyanin did yield a conjugate with at least 6.h. and probably over 20. molecules of hapten attached to the carrier protein molecule. Through an adjuvant assisted immunization and periodic booster enhancement. the rabbits formed antibodies to both hemocyanin and to the epoxy FA hapten. This was quantified and periodically determined to plot the decline from a peak 22.1 ug/ml after the last booster. to h.6 ug/ml 5% months later. The plotted decline was consistent with the general immunological predictions and further. yields original data on how this specific small hapten responds in rabbits. The application of anti epoxy FA serum to indirect fluorescent antibody staining of adult cockroach corpra allata tissues and hemolymph smears produces specific fluorescence. Most likely. this specific fluorescence is localized at the sites of endogenous juvenile hormone and/or its metabolic precursors with 72 "J 73 very similar molecular configurations. Explanation of the observed increase of hemocyte fluorescence intensity over that of the corpra allata tissues awaits further investigation. There are several possibilities worthy of consideration. the T13 first being that much of the juvenile hormone material ‘ is in the form of a storage precursor molecule which i does not cross-react with the antibody. The second possible area to investigate would be the relationship A of the JH in association with carrier lipoproteins in the hemolymph. and membrane surface association with the hemocytes. Either of these would contribute to the physical availability of JH to the antibody or might provide a more stable surface for attachment and resultant higher survival of the antibody antigen complex through the several washings. BIBLIOGRAPHY Ajami. A. M. and Riddiford. L. M.. 1973. Comparative metabolism of the Cecropia juvenile hormone. {—1 J. Insect Physiol. ‘12: 635-646. T Bartels. P. Fluorescence Microscopy: Principles. Applications and Instrumentation. E. Le tz. Inc.. Publ. .‘v_ W, A‘q‘M vqiiglig ' an.“ .. Bassi. S. D. and Feir. D.. 1971. 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